Extended Data Fig. 6: Workflow of subtomogram averaging of HIV-1 WT CA hexamers and NPC and NPC subunit symmetry analysis.

a, The workflow used in this study for subtomogram averaging of CA hexamers. A low-pass filtered (40 Å) template was applied for the initial template matching using emClarity/1.5.0.2. The peak number was intentionally set to an excessive value in cropped tomograms to ensure an exhausting search. The matched particles were initially cleaned using MagpiEM, followed by manual inspection in Chimera to further remove false positives. The cleaned particles were then transferred into RELION/4.0 for 3D classification, good classes are selected for the following iterative refinement. b, Gold-standard Fourier shell correlation (FSC) curves of subtomogram averaged maps from imported (light purple), traversing (purple), and outside (approaching and docking combined, light blue) CA hexamers. The resolution is indicated at 0.143 FSC cut-off. c, The workflow for symmetry analysis of NPCs in this study. The initial NPC structure (EMD-11967) was low-pass filtered to 60 Å and cropped to 1/8^th of the original volume based on the 8-fold symmetry. The cropped map was then applied for the template matching via emClarity. The matched particles were initially cleaned by MagpiEM and then inspected in Chimera based on the cross-correlation value and orientation for further cleaning. The cleaned particles were then mapped back into the tomogram for the calculation of included angles between adjacent NPC subunits and iterative refinement in RELION/4.0. d, Gold-standard Fourier shell correlation (FSC) curves of subtomogram averaged maps from NPC CR (blue), IR (cyan), LR (green), and NR (black). The resolution is indicated at 0.143 FSC cut-off.